1. Field of the Invention
The present invention relates generally to chemical agents useful in the prophylaxis and/or treatment of disease conditions and in particular chronic disease conditions such as inflammatory including allergic diseases, metastatic cancers and infection by pathogenic agents including bacteria, viruses or parasites. More particularly, the chemical agents contemplated by the present invention are selected from glycosaminoglycan (GAG) molecules derived from a larger GAG, GAG-like molecules which resemble GAGs in some of their characteristics but may be derived from a larger non-GAG polysaccharide and molecules having a GAG-like composite structure as well as agents which bind to the same sites as GAGs, GAG-like molecules or GAG-like composite molecules. The present invention also provides assays to identify GAG and GAG-like therapeutic agents including GAG-like composite structures as well as analogs, homologs and orthologs thereof.
2. Description of the Prior Art
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
The development of many disease conditions in a host involves the interaction between cell or viral entities or molecules produced thereby and cells in the particular host. One well studied interaction is between HIV, gp120 and lymphocyte CD4 receptors [Eckert and Kim, Annu. Rev. Biochem. 70: 777-810, 2001]. Despite some success, however, in antagonizing or facilitating these interactions, it has been difficult to identify an antagonist that exhibits a sufficiently broad spectrum of activity.
In work leading up to the present invention, the inventors investigated heparin-like glycosaminoglycans (HLGAGs) such as heparin and heparan sulfate and the oligosaccharides derived from them. Glycosaminoglycans (GAGs) are ubiquitous and play pivotal roles in many of the inflammatory processes within the human body. These large molecular weight polysaccharides contribute to such processes as cancer metastasis, arthritis, transplant rejection, allergic rhinitis and asthma and thus a greater understanding of these processes leads to improved drugs for the treatment of such conditions. Currently, one of the best known GAGs is the heparin family of sulfated polysaccharides and the anti-coagulant activity of these molecules is well understood.
However, HLGAGs are a heterogenous group of molecules [Conrad, Heparin binding proteins. Academic Press, San Diego, 1998; Lander and Selleck, J. Cell Biol. 148(2): 227-232, 2000]. Heparin and heparan sulfate, like all HLGAGs, are long linear polysaccharides [Sasisekharan and Venkataraman, Curr. Opin. Cliem. Biol. 4(6): 626-631, 2000; Casu, Ann. N.Y. Acad. Sci. 556: 1-17, 1989; Casu, Adv. Carbohydr. Chem. Biochem. 43: 51-134, 1985]. They are synthesized as non-sulfated chains of repeating disaccharide units comprising glucuronic acid (GlcA) and glucosamine (GlcN) which, in the golgi, are modified at various sites along their length. Heparin is more extensively modified than heparan sulfate and most of the GlcN units are modified by a sulfate group to become N-sulfated GlcN and most GlcA units are converted to iduronic acid (IdoA) through the action of epimerase. HLGAGs are heterogenous since modifications to the sulfate chains are often incomplete. The result is extensive regions of intermediate modification.
Thus, for example, heparan sulfate chains consist of highly sulfated, structurally flexible domains rich in 2-O-sulfated IdoA alternating with regions of low sulfation consisting predominantly of N-acetyl GlcN and GlcA, which are a rigid structure.
The sulfation patterns of HLGAGs are complex especially with respect to the positioning of 6-O-sulfates. Consequently, not all HLGAG molecules are identical. Similarly, not all molecules in a preparation of HLGAGs from a particular cell or tissue are identical; rather such preparations represent a family of molecules.
It is the sulfation pattern which largely determines the protein binding characteristics of a particular HLGAG. Some proteins bind only to particular structural motifs within a HLGAG chain and conversely some GAGs bind only to particular sites or regions on a protein. Anti-thrombin III, for example, binds to a unique pentasaccharide sequence displaying a particular arrangement of sulfate groups and the heparin pentasaccharide binds to a specific site on the anti-thrombin III protein [Whisstock et al., J. Mol. Biol. 301: 1287-1305, 2000]. Basic fibroblast-derived growth factor (FGF-2) and hepatocyte growth factor (HGF) both bind heparin, but the heparin structures that are essential for binding are quite different for each and are different from that required by anti-thrombin III [Maccarana et al., J. Biol. Chem. 268(32): 23898-23905, 1993; Lyon et al., J. Biol. Chem. 269: 11216-11223, 1994]. Moreover, heparin has been shown to bind to a particular region on FGF-2 [Faham et al., Science 271: 1116-1120, 1996]. The binding of sulfated GAGs and proteins in the anti-coagulation cascade is very complex and the interaction of heparin with platelet factor IV (PF-IV) is something that has been problematic in some therapeutic applications. Elegant work has shown that, although the heparin-PF-IV is an extremely high affinity interaction, the specificity is also high, thus by adding functionality that is non-detrimental to heparin binding and yet detrimental to PF-IV binding, some selectivity may be achieved (Petitou et al., Nature 398: 417, 1999.) This was achieved by minimizing the number of highly sulfated regions to 4-5 saccharide units on the termini and having a non-charged oligosaccharide spacer separating the charged sections.
FGF-2 recognizes a motif containing a single IdoA 2-O-sulfate in a defined position, whereas for HGF, the positioning of the GlcN 6-O-sulfate groups are critical. Some heparin molecules within a preparation will carry both the anti-thrombin III binding pentasaccharide and the FGF-2 binding motif, whereas others will carry the HGF binding motif and the FGF-2 motif and not the anti-thrombin III binding pentasaccharide. Indeed, on average only one third of the molecules within a preparation of heparin carry the anti-thrombin III binding pentasaccharide [Conrad, 1998, Supra].
Thus, the molecules in a preparation of HLGAGs differ in the order, in the number, and in the types of protein binding sites. The challenge is to identify, in structural terms, the HLGAG motif that binds the protein of interest and the HLGAG binding site on the protein. The isolation and purification of that HLGAG motif should give a reagent that is potent and more specific in its binding behaviour. Once the carbohydrate structure of the motif is known, it is then possible to make structural analogs or orthologs (i.e. functionally equivalent structures) that may or may not contain GAGs as part of their structure and which can be clinically used.
The heterogeneity present in the heparin isolated from natural sources (mast cells of pigs) gives rise to many side effects. The search for a GAG, or a structural analogue, without, or with reduced anti-coagulant activity could pave the way for more effective treatments of the inflammatory conditions outlined above.
Sources of GAG polysaccharides include from natural sources, as well as synthetic or semi-synthetic sources.
Many “natural” type glycosidic linkages predominate as a function of various enzymatic systems that are responsible for the biosynthesis of these oligosaccharides. Accessing quantities of these complex oligosaccharides from natural sources necessitates repeated purification by chromatography and due to the similarity of many of these oligosaccharides, homogeneous samples are difficult to obtain.
The capsular polysaccharide from E. coli K5 is composed of an alternate α-N-acetyl glucosamine (α-GlcNAc) and β-glucuronic acid (β-GlcA) units and contains no sulfate or other charged groups (see FIG. 17). The heparin backbone consists of the following motif α-GlcNAc, β-GlcA, α-GlcNAc, β-iduronic acid (β-IdoA) with varying degrees of sulfation. The only difference between GlcA and IdoA is the configuration of the carboxylic acid group at C-5, thus the heparin backbone and the E. coli K5 backbone are extremely similar in structure (FIG. 17). Indeed the K5 polysaccharide displays very low immunogenicity. Escherichia coli K5 is capable of producing approximately 50 mg/L from a growth medium and thus is ideally suited for the multi-gram production of the desired heparin-like backbone.
Suitable strains of E. coli for the production of K5 polysaccharide are NCDC Bi 626-42 and NCDC Bi 8337-41 and both are available from the American Type Culture Collection (ATCC). A number of publications describe isolation of the K5 polysaccharide. See, for example, Leali et al., J. Biol. Chem. 276: 37900-37908, 2001; and Finke et al., J. Bacteriol. 173: 4088-4094, 1991. Briefly, a medium rich in glycerol in preference to glucose is used to grow the bacteria. Conditioned media is collected from late logarithmic phase cultures and concentrated using a 10,000 Da cut-off ultrafiltration membrane. The polysaccharide is recovered by acetone precipitation, any proteins are digested with protease II and ultrafiltration and precipitation is used to recover the polysaccharide. Some methods also suggest an anion exchange chromatography step (U.S. Pat. No. 5,341,876).
In accordance with the present invention, GAG and GAG-like molecules including GAG-like composite molecules are identified which interact with target ligands such as cytokines or growth factors. Such molecules, including their chemical analogs, homologs and orthologs, are proposed to have useful therapeutic or diagnostic properties.